| Fungal sexual reproduction has become the focus of many scholars because of its diversified characteristics and important roles in biological evolution,gender origin,species differentiation and genetic inheritance variation.In ascomycetes,sexual reproduction is controlled by the mating type genes,which not only control sex differentiation and sexual development,but regulate the formation of ascus shells,etc.,which play a decisive role in the genetic evolution and sex control of fungi.Because of their diverse reproductive patterns,the fungi of Orbiliaceae have become an ideal material to study the sexual reproduction mechanism of fungi.In this paper,based on the Orbiliaceae fungi isolated and preserved,the mating type locus is used as the research object,and the model species Arthrobotrys oligospora is taken as an example,using homology comparison,gene knockout,phenotypic experiment and transcriptome analysis to make preliminary explorations for structure and function of mating type genes and population genetic structure.This study provides a valuable reference for further understanding the mechanism of sexual reproduction and the role of mating type genes in Orbiliaceae fungi.The main experimental results are as follows:1.The MAT gene was predicted and annotated on the basis of the whole genomes of ten strains of six genera of the Orbiliaceae.Among them,Drechslerella brochopaga（YMF1.03216）had the largest MAT locus,which was 154177 bp,and contained 41 Genes.Dicranidion sp.（YMF1.03024）had the smallest MAT locus,which was 14286 bp and contained 2 genes,MAT1-1-1 and MAT1-2-1 genes.Except for Orbilia laevimarginata（YMF1.01833）,the upstream of the MAT locus of other strains was that APN2 was closely connected with the MAT locus and Cox13,respectively,while the downstream of the MAT locus had large mutations,and the genes were reversed.In addition,the structural order of MAT loci within species was relatively conservative,but there were obvious differences between species.2.We used A.oligospora of the genus Arthrospora of the Orbiliaceae family as the experimental strain,and useed homologous recombination to knock out its genes to obtain knockout strains of MAT1-1-1 and MAT1-2-1 gene.Phenotypic experiments were performed on wild strains and corresponding knockout strains.Results demonstrated that the growth velocity of ΔAo MAT1 was notably higher than that of the wild strain on the four media,while the growth rate of ΔAo MAT2 was different from that of the wild strain.In the comparison of stress resistance,the ΔAo MAT1 and ΔAo MAT2 knockout strains had different experimental results in different stress environments compared with the corresponding wild strains,but the results of the two were opposite,which may indicate MAT1-1-1 and MAT1-2-1 genes were different in regulating the growth of A.oligospora and adapting to adversity environment.Comparison of sporulation showed that MAT1-1-1 and MAT1-2-1 genes may be involved in the regulation of sporulation and conidia production,and the regulation mechanisms were opposite.The results of comparison of nematocidal activity and trap production showed that the number of traps of ΔAo MAT1 was lower than that of the wild strain at first,and was notably higher than that of the wild strain at 36 h and 48 h with the extension of time,while the number of trap of ΔAo MAT2 knockout strain was always higher than that of the wild strain,indicating that the regulation of sporulation and trap production of MAT1-1-1 was more complex than that of MAT1-2-1.3.In order to furtherly investigate the regulatory role of MAT1-1-1 and MAT1-2-1genes in the interaction between A.oligospora and nematodes at different growth stages,according to the results of nematocidal activity and trap production,the samples of knockout strains and wild-type hyphae interacting with nematodes at four time points of0 h,12 h,24 h and 36 h were collected for transcriptome sequencing,and the data were screened for differential genes and analyzed by GO and KEGG Pathway enrichment.We found that MAT1-1-1 was mainly involved in the biogenesis of ribosomes and the regulation of proteasome.MAT1-2-1 was mainly involved in the regulation of various amino acid metabolism and degradation pathways,DNA replication and mismatch repair,biosynthesis,meiosis,cell cycle and sucrose metabolism of A.oligospora.4.In order to investigate the relationship between the genetic variation of the MAT genes and the ecological adaptation of A.oligospora,200 strains of A.oligospora were collected,isolated and identified across the country as experimental objects.We obtained85 strains with MAT1-1-1,115 strains with MAT1-2-1,and divided them into two groups for genotype and population genetic analysis.The results showed that MAT1-1-1had 15 genotypes and 101 SNPs（multiple nucleic acids）,the region with the most genotypes was Gejiu,Yunnan.MAT1-2-1 had 13 genotypes and 133 SNPs,and the genotype was Guangxi at most.The results of AMOVA analysis showed that both intraand inter-population of MAT1-1-1 and MAT1-2-1 contributed to genetic differentiation,and both groups were significant.Mantel test showed that there was a positive correlation between genetic distance and geographical distance between MAT1-1-1populations,while there was a negative correlation between genetic distance and geographical distance between MAT1-2-1 populations. |
